In article <jmanalig-2009950944170001 at host40-44.oto.uiowa.edu>,
jmanalig at blue.weeg.uiowa.edu (jose) wrote:
> I've been toying with the idea of hot start PCR to get a cleaner PCR
> product and minimizing primer mismatch. However, by looking at the
> literature and the catalogs, there seems to be a few different ways in
> which to do this ranging from using wax to separate Taq polymerase from
> your other reagents, using wax beads with Mg and a Mg free buffer etc.
> What's the best and/or easiest way to do this?
>> --
> Jose Manaligod, MD
> University of Iowa Hospitals and Clinics
> Iowa City Iowa
>jmanalig at blue.weeg.uiowa.edu
>In my opinion, Hot Start PCR is a total waste of time. I use a protocol
>called "touchdown PCR" which increases specificity without having to stop
>and add stuff. (When you're doing 320 reactions a day, this is a big deal)
>You just start off with an annealing temp that's about ten degrees
>higher, and then decrease 1 degree every cycle to your optimum temp.
>Finish the rest of the cycles at that temp. I choose all my primers to
>have an annealing temp of about 60 degrees, and use the same touchdown
>program every time.
>beth
The simplest Hot Start is setting your samples at ~80°C and then adding
your polymerase and then cycling, no expense and it works. If this does
improve your amplification specificity and you do too many reactions to do
it manually then one of the other methods could be used. Wax is effective
and cheap, you needn't but the $1 per bead PCR Gems from PE, Aldrich sells
~1kg for $20. Heat the wax and drop onto teflon, or into DI H2O, about
20ul, but exp to see what works for your tubes & reaction volumes, you can
also drop the wax into waiting tubes on the side as some vendors now
do.Once the wax is melted on top of the mix, cooling the wax shouldn't
leave a open hole in its center. Make sure the top layer is large enough
to cause effective mixing when the wax melt (~1/2 vol). Going through the
wax after the run is a pain, I always pierce with one tip and withdraw
with another.
Beths procedure may work for her through put but if I'm doing two temp
PCR and/or want more than ten cycles (-1° per cycle?) its best to do a Hot
Start. Touchdown and Hot Start are not mutually exclusive and in really
hairy cases may be necessary to perform in concert. The Mg in the beads is
really just a gimmick, are the odds greater you have stray Taq pol around
or stray Mg? The TaqStart antibody is a good idea as it should allow Hot
Start to be used in sealed capillaries for even greater specificity, but
it is , as stated earlier, expensive. ( I wonder if the antibody would be
useful in Taq pol production/purification, that would offset the cost a
bit...)
My bottom line is that if you need better specificity start with the
easy exp's:
1. raise anneal temp until your product goes away and/or you lose the
nonspecific priming. I've seen 18mers prime beautifully at 72°C,
>15° over their calculated Tm!!
2. Try a manual Hot Start and if it works you can move to a barrier method.
3. try capillary PCR/ Touch Down, fewer cycles, low dNTPs, etc etc etc.
Good luck.
regards,
Ted Michelini
Institute of Molecular Biology
University of Oregon
tedm at darkwing.uoregon.edu